For many years, agar-filled pour plates provided the best method of determining the number of microorganisms in a liquid sample, such as water or milk. However, the use of agar medium is particularly inconvenient and time-consuming. For example, agar medium must be sterilized, melted, and cooled prior to addition of the liquid sample. Furthermore, the sample and medium must be mixed, solidified, and incubated prior to counting of the number of microorganism colonies which grow on the plate.
To date, the prior art has provided several devices useful for assaying liquid specimens for microorganisms which are easier and quicker to use than traditional agar pour-plate technology. For example, German patent application No. 2055741, published May 19, 1971, discloses a microbiological growth medium comprised of an inert card or strip coated or impregnated with a dry-gelled nutritive medium. In one embodiment, the inert card or strip may include optional side walls to prevent shifting of the medium after wetting with a liquid sample. In addition, the nutritive medium may include an adhesive component or be adhered to the card or strip by an intervening adhesive layer. Furthermore, an optional sponge material can be disposed between the card or strip and the nutritive medium, and the nutritive medium can be covered by a semi-permeable membrane.
U.S. Pat. No. 4,565,783 (assigned to the Assignee of the present invention) provides a culture media device comprised of a dry-powdered gelling agent and/or nutrient composition adhered to a waterproof substrate by a layer of water-insoluble adhesive which is non-inhibitory to the growth of microorganisms. Upon application of a liquid sample to the device, the gelling agent(s) hydrates to form a gelatinous medium useful for growing microorganisms contained in the liquid sample. In addition, the device can also include a transparent cover sheet and/or a hydrophobic spacer element with side walls to maintain a pre-determined amount of a liquid sample in contact with the dry-powdered gelling agent(s) and/or nutrient composition of the culture media device. Any nutritive components and/or other agents are incorporated along with the gelling agent(s) into the dry-powdered media coating the device. Alternatively, the nutritive component and/or other agents can be incorporated into a substantially water-free, non-adhesive composition coated onto the waterproof substrate. However, dry-powdered gelling agents cannot be utilized to coat such an embodiment. Commercial embodiments of such devices include Petrifilm.TM. brand growth media, available from 3M, St. Paul, Minn.
European patent application No. 0374905, published Jun. 27, 1990, also discloses a device for culturing microorganisms comprised of a base sheet composed of a lower water repellent sheet and an upper hydrophilic sheet, such as filter paper. A gel agent or gelatinizer is dispersed in the upper hydrophilic sheet and then solidified. Thereafter, a water-repellent sheet is applied to cover the upper surface of the hydrophilic upper sheet.
U.S. Pat. application Ser. No. 07/354,627, allowed on Sep. 16, 1991, now U.S. Pat. No. 5,089,413, and assigned to the Assignee of the present invention, provides yet another microbiological dry culture medium device. The device is constructed in an analogous fashion to the culture media device of U.S. Pat. No. 4,565,783, described above, except that the base of the device is comprised of an air-permeable membrane adhered to the upper surface of the waterproof substrate. Utilization of the air-permeable membrane provides a means for growing oxygen dependent microorganisms, such as molds, even when an air-impermeable cover sheet is placed over the inoculated culture medium.
The above-described devices have not addressed several areas that are important to the successful construction and use of culture media devices. For example, many conventional adhesives inhibit microorganism growth due to their strongly anionic or cationic nature, or through the deliberate incorporation of antimicrobial agents. Thus, culture devices that incorporate such an adhesive component in, or adjacent to, the nutritive medium may inhibit rather than facilitate the growth of microorganisms.
Even when non-inhibitory adhesives are utilized, such as in U.S. Pat. No. 4,565,783, the water-insoluble nature of the adhesive renders them essentially incapable of holding all but the smallest quantities of water-soluble nutrients and/or other hydrophilic agents. Thus, these nutrients and/or other agents must be incorporated into other 20 water-soluble layers, such as the substantially water-free, cold-water-reconstitutable material of U.S. Pat. No. 4,565,783, and/or various other dry-powdered media. However, these forms of media typically may not provide for adequate control on the release rates of the nutrients and/or other agents. Furthermore, concentration gradients of these components also can occur when these dry media are hydrated.
The lack of control of release rates and creation of concentration gradients is of particular concern with inhibitory agents. Rapid and high dosage release of inhibitory agents may in fact lead to non-selective inhibition of microorganism growth. In addition, creation of concentration gradients of such inhibitory agents may lead to growth inhibition on one portion of the device, but not on another. Under either scenario, the ability to grow and accurately quantify microorganism colony growth is lost.
Finally, utilization of water-absorbing elements, such as sponges and/or filter paper, may not sufficiently contain colony growth, thereby limiting the quantitative value of such devices, and making microorganism colony isolation impractical. In addition, neither of these structures are sufficiently transparent to allow for the counting of colonies through the substrate, thereby also rendering the accurate counting of microorganism colonies nearly impossible.